Histone Code Modifications on Pluripotential Nuclei of Reprogrammed Somatic Cells

Abstract

Following hybridization with embryonic stem (ES) cells, somatic genomes are epigenetically reprogrammed and acquire pluripotency. This results in the transcription of somatic genome-derived tissue-specific genes upon differentiation. During nuclear reprogramming, it is expected that DNA and chromatin modifications, believed to function in cell-type-specific epigenotype memory, should be significantly modified. Indeed, current evidence indicates that acetylation and methylation of histone H3 and H4 amino termini play a major role in the regulation of gene activity through the modulation of chromatin conformation. Here, we show that the reprogrammed somatic genome of ES hybrid cells becomes hyperacetylated at H3 and H4, while lysine 4 (K4) of H3 becomes globally hyper-di-and -tri-methylated. In the Oct4 promoter region, histones H3 and H4 are acetylated and H3-K4 is highly tri-methylated on both the ES and reprogrammed somatic genomes, which correlates with gene activation and DNA demethylation. However, H3-K4 is also di-and tri-methylated in the promoter regions of Neurofilament-M (Nfm), Nfl, and Thy-1, which are all silent in both ES and hybrid cells. Thus, H3-K4 di-and tri-methylation of reprogrammed somatic genomes is independent of gene activity and represents one of the major events that occurs during somatic genome reprogramming towards a transcriptional activation-permissive state.

Epigenetic Reprogramming ofOCT4andNANOGRegulatory Regions by Embryonal Carcinoma Cell Extract

“…This takes place in the context of minimal hypermethylation of already di-and trimethylated H3K4, which mark genes either transcriptionally active (H3K4m3) or competent for transcription (H3K4m2) (Santos-Rosa et al, 2002). These changes are consistent with chromatin remodeling on the Oct4 promoter in mouse thymocyte-ES cell hybrids, except for the marked trimethylation of (initially unmethylated) H3K4 detected in the thymocyte nuclei (Kimura et al, 2004). Transcriptional activation of Oct4 in mouse fibroblasts treated with ES cell extract requires ATP hydrolysis, most likely for nuclear import of transcription factors Landsverk et al, 2002) and for the activity of chromatin remodeling complexes (Kingston and Narlikar, 1999;Aalfs et al, 2001).…”

“…Partial DNA demethylation in restricted areas in the Oct4/OCT4 regulatory region has been reported previously (Tada et al, 1997;Simonsson and Gurdon, 2004;Cowan et al, 2005;Takahashi and Yamanaka, 2006), and it is proposed be required for activation of the gene (Simonsson and Gurdon, 2004). These studies have been extended with the demonstration that the Nanog promoter is also demethylated in nuclear transfer ES cells (Blelloch et al, 2006), in fibroblast-ES cell hybrids (Cowan et al, 2005), and in transduced cells (Takahashi and Yamanaka, 2006 thymocyte-ES cell hybrids (Kimura et al, 2004). A limitation of cell fusion or transduction approaches to nuclear reprogramming, however, is the mixing of genomic sequences, making epigenetic analyses of the reprogrammed cells dependent on single nucleotide polymorphism or species specificity of the sequences examined.…”

“…To support this view, these modifications were not as prominent as in NCCIT cells, in which both genes are transcribed at a higher level than in reprogrammed cells. Notably, H3K27m3 seems to be fully demethylated in thymocyte-ES cell hybrids, suggesting a more extensive chromatin remodeling in this system (Kimura et al, 2004). Globally, however, the changes reported as a result of EC extract treatment reflect a remodeling of chromatin on OCT4 and NANOG indicative of a transition from a potentially active to an active promoter.…”

“…These studies have been extended with the demonstration that the Nanog promoter is also demethylated in nuclear transfer ES cells (Blelloch et al, 2006), in fibroblast-ES cell hybrids (Cowan et al, 2005), and in transduced cells (Takahashi and Yamanaka, 2006). Additionally, acetylation and methylation of lysine (K) residues in the aminoterminal tail of histones H3 and H4, which regulate chromatin assembly on promoters and thereby promoter activation (Lachner and Jenuwein, 2002), have been shown in mouse thymocyte-ES cell hybrids (Kimura et al, 2004). A limitation of cell fusion or transduction approaches to nuclear reprogramming, however, is the mixing of genomic sequences, making epigenetic analyses of the reprogrammed cells dependent on single nucleotide polymorphism or species specificity of the sequences examined.…”

Mechanisms of nuclear reprogramming by eggs and oocytes: a deterministic process?

“…1d). In the other, the chromosomes of the fused cells assemble together on a shared mitotic spindle and form a fused nucleus (a synkaryon) 13 . Similarly to egg-NT, reprogramming in synkaryons is hard to analyse because of the cell divisions that follow the fusion of nuclei and cytoplasms.…”